ABSTRACT Although cancer immunotherapy is considered a great breakthrough in cancer therapy, a significant proportion of patients do not respond to these treatments. Recent studies have shown that clinical benefits of the most potent immunotherapy lay in the presence of a T cell inflamed tumor microenvironment at baseline (hot tumor). Dysregulated Wnt/?-catenin signaling has been implicated in the lack of immune cell infiltration in tumors and underlies the pathogenesis of a broad range of human cancers. Specifically, upon association with cofactor and onco-gene Bcl9, ?-catenin promotes transcription of genes associated with stemness, proliferation, survival and metastasis. Over expression or mutations in Bcl9 or ?-catenin have been associated with tumor progression, decreased survival, and poor clinical outcome in a broad range of human cancers including colorectal cancer. In addition, recent studies in mouse melanoma tumor models have shown that cell intrinsic ?-catenin signaling plays a critical role in evasion of immuno-surveillance. Both Bcl9 and ?-catenin expression have been shown to correlate with poor immune cell infiltration and survival in metastatic melanoma and 52% of patients with non-T cell inflamed melanoma lesion show evidence of activation of the Wnt/?-catenin pathway. Thus, this pathway is hypothesized to be an important target to restore immune infiltration in tumors. Moreover, Bcl9/?-catenin modu- lation is hypothesized to be synergistic with treatment modalities that boost the anti-tumor immune response. We have developed a series of nuclear ?-catenin inhibitors that selectively interfere with Bcl-9/?-catenin interac- tions and hold the promise of mediating the Wnt/?-catenin pathway towards restoring anti-tumor T-cell activity, while avoiding toxic effects associated with ?-catenin inhibition or disruption of other routine non-immunological activities of ?-catenin. Our three preliminary lead compounds VTX-003a, b and c, showed a significant capacity to specifically inhibit nuclear BCL9/?-catenin interactions and the subsequent induction of cancer associated gene transcription in two human colon carcinoma cell lines. Furthermore, these compounds have demonstrated promising ADME and pharmacokinetic (PK) profiles in mice in vivo upon oral administration. We propose to expand on our lead series of selective nuclear ?-catenin inhibitors, as well as evaluate and de- termine the mechanistic and efficacy properties of our compounds on Wnt/?-catenin signaling and immune-ex- clusion in colorectal cancer (CRC) models, towards advancing a ??catenin inhibitor capable of turning these tumors hot and increasing the viability of cancer immunotherapy. We first propose to utilize a scalable and mod- ular synthetic route for rapid generation of analogs, which we have developed, to readily access a vast number of analogs and rapidly assess not only their activity in our initial ??catenin assays but also evaluate their ADME and PK characteristics (Specific Aim #1). Then, we will characterize our leads in vitro (Specific Aim #2). Finally, we will test for the efficacy of VTX-003c and yielded analogs that emerge from Specific Aim #1 in murine synge- neic colon carcinoma transplant models (Specific Aim #3).